Limited proteolysis of inactive precursors to produce active peptides and proteins is an ancient mechanism to generate biologically diverse products from a finite set of genes. Most often, such processing occurs at either single or dibasic residues, as a result of cleavage by a family of mammalian serine proteinases related to bacterial subtilisin and yeast kexin(1, 2). These enzymes, known as pro-protein convertases (PCs), participate in the tissue-specific intracellular processing of precursors at the consensus (R/K)−(X)n—R↓ sequence, where X is any amino acid except Cys and n=0, 2, 4 or 6 (1–3). PCs have been implicated in the production of various bioactive polypeptide hormones, neuropeptides, enzymes, growth factors, adhesion molecules, cell surface receptors and surface glycoproteins of infectious agents such as viruses and bacteria (1–3).
Less commonly, bioactive products can also be produced by limited proteolysis at amino acids such as Leu, Val, Met, Ala, Thr, Ser and combinations thereof (3). This type of cellular processing has been implicated in the generation of bioactive peptides such as α-and γ-endorphin (4), the C-terminal glycopeptide fragment 1–19 of pro-vasopressin (5), anti-angiogenic polypeptides such as platelet factor 4 (6) and angiostatin (7), the metalloprotease ADAM-10 (8), site 1 cleavage of the sterol receptor element binding proteins (9), as well as in the production of the Alzheimer's amyloidogenic peptides Aβ40, 42 and 43 (10). Processing of this type occurs in the endoplasmic reticulum (ER) (9), or late along the secretory pathway, within secretory granules (4, 5), at the cell surface, or in endosomes (6–8, 10). So far, the proteinases responsible for these cleavages have not been unambiguously identified.
Since mammalian convertases process precursors at either single or pairs of basic residues, we hypothesised that a distinct, but related, enzyme(s) may generate polypeptides by cleavage at non-basic residues. To test that idea, we employed an RT-PCR strategy similar to the one used to identify the PCs (11), except that we used degenerate oligonucleotides closer to bacterial subtilisin than to yeast kexin. This approach resulted in the isolation of a cDNA fragment encoding a putative subtilisin-like enzyme from human cell lines. This partial sequence was identical to a segment of a human myeloid cells-derived cDNA reported by Nagase et al. (12). A role for this putative subtilase remained undefined up to the present invention.
It was further discovered by Cheng, D. et al. (1999) J. Biol. Chem. 274.22805-22812 that an enzyme call S1p, is capable of cleaving sterol-regulatory element-binding proteins (SREBPs), which function to control lipid biosynthesis and uptake in animal cells. Upon cleavage, SREBPs are released from cell membranes for translocation to the nucleus, where they activate transcription of genes involved in the biosynthesis and uptake of cholesterol and fatty acids. S1p and the present enzyme or the same. Therefore, for diseases involving overexpression of these genes as well as any other disease involving SKI-i activity, it is contemplated that any inhibitor of SKI-1 would be useful in their treatment